1. Utilizing NGS-Data to Evaluate Anti-PD-1 Treatment
Vera Varis
Fall 2018

2. PD-L1 and PD-1 PD-1 inhibits CTLs (Cytotoxic T-Lymphocytes)
Pembrolizumab
PD-1 inhibits CTLs (Cytotoxic T-Lymphocytes)
Inhibition leads to defects in T-cell expansion
Defects in T-cell response to tumors
Picture:
Cellular and Molecular Immunology 9th Ed.
Abbas A. et. al. Elsevier (2017)

3. Forward Genetics
Genotype
Phenotype
Hypothesis
Test hypothesis

4. Anti-PD-1 Treatment Studies
Sequencing (WES/RNA-seq)
Transcriptomics
Healthy tissue
Genotype
Data analysis
Phenotype
Hypothesis
Tumor samples
Differences between samples:
Genomic
Transcriptomic/ expression patterns
Post-treatment
Test hypothesis
Pre-treatment
Anti-PD-1 treatment
Combinatory treatments

5. Response to Anti-PD-1 Treatment: Genomics and Transcriptomics
Goals to identify genomic features contributing to anti-PD-1 response
Strategies for patient stratification
Combintory therapies
Whole-exome sequences and transcriptomes of 38 pre-treatment melanoma tumors and patient matched normal tumors
HUGO, Willy, et al. (2016). Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell, 165.1:

6. Detection of non-synonymous somatic mutations (nsSNV)
Mutational Loads
Detection of non-synonymous somatic mutations (nsSNV)
Higher mutational load was strongly associated with improved survival
High mutational load does not correlate with tumor response to PD-1 blockade

7. Mutations in BRCA2 nsSNV counts for BRCA2: Non-responding 1/17 (6%)
Loss-of-function mutations
Somatic mutational load higher in
BRCA2 mutants compared to the WT
BRCA2 mutations enriched in anti-PD-1 responsive melanoma

8. Transcriptomic Signatures
Differentially expressed genes (DEGs)
Responding vs. non-responding
693 genes differentially expressed
Gene up-expression in non-responding tumors
GO-analysis: non-responding tumors

9. Transcriptomic Signatures
Analysis of molecular and cellular processes in non-responding vs. responding tumors
Independently derived perturbation-based transcriptomic signatures
Anti-PD-responsive group did not have higher enrichment of IFN-signatures
Co-enrichment of 26 transcriptomic signatures
Non-responding 9 of 13
Responding 1 of 15
Innate anti-PD-1 resistance signature (IPRES)

10. Conclusion: IPRES Signatures and Importance of Exome and Transcriptome Sequencing
Anti-PD-1 non-responding pretreatment tumors had high enrichment of IPRES signatures
IPRES signatures were enriched in all major common human malignancies
Comparison of IPRES-enriched subsets
Combinatory treatments?
MAPKi
Anti-CTLA-4
IPRES
IPRES
Mesenchymal transition
ECM remodeling
Hypoxia
Angiogenesis
= CTL

11. PD-1 Blockade and Mismatch Repair Deficiency
Evaluation of anti-PD-1 treatment
86 patients
12 different tumor types
Mismatch-repair deficient cancers
Primary tumors vs. metastases
ns somatic mutations
Does checkpoint blockade induce peripheral expansion of tumor-specific T-cells?
Sequencing of TCR Vβ CDR3 regions
Do mismatch repair specific tumors harbor functional MANA-specific T-cells?
MANA = mutation-associated neoantigen
Reference:
Le, D. T., Durham, J. N., Smith, K. N., Wang, H., Bartlett, B. R., Aulakh, L. K., ... & Wong, F. (2017). Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science, 357(6349),

12. WES-Data of Biopsies of Brain Metastases
Two individuals
Comparison of mutations in metastases vs. primary tumors
Qualitative (in figure) and quantitative study of mutations
Conclusion:
Metastases derived from the tested primary tumors
Both brain metastases had mutations in BM2 gene
Non-synonymous somatic mutations

13. Affects of PD-1 Blockade to Tumor Specific T-cell Expansion
Sequencing of TCR Vβ CDR3 regions (TCR-seq)
Tumors from three patients responsive for pembrolizumab
Identification of intratumoral clones and their peripheral expansion
All three samples:
Very low frequency of clones pre-treatment
Increase post-treatment, followed by contraction
Picture:
Cellular and Molecular Immunology 9th Ed. Abbas A. et. al. Elsevier (2017)
Patient 19 chosen for further studies

14. TCR-seq: Expanded Lymphocyte Population Against Seven Peptides
Patient 19:
Post-treatment peripheral blood tested for 15 candidate MANAs
T-cell responses for 7 of 15 MANAs found based on counts of spot-forming cells or cytokine activity analysis
TCR-seq and expanded lymphocyte populations against the 7 peptides
Result:
T-cell expansion in response to three peptides
Expansion resulted in 142 unique TCR sequences
7 of which are found in tumor samples
Frameshift mutations

15. MANA-Specific Clones
All seven of the MANA-reactive TCR:s detectable in peripheral blood pre-treatment
Frequency of four T-cell clones increased rapidly after anti-PD-1 treatment
Frequency decreased soon after T-cell clonal expansion
Similar pattern as in acute viral infections
(not shown here)

16. The Importance of Mismatch Repair Deficiency
Mismatch Repair Deficiency Across 12,019 Tumors
Identification of patients resposive to PD-1 blockade
Not only melanoma – also other tumor types
Annual diagnoses in the US:
40,000 for stages I-III
20,000 for stage IV

17. Conclusion and Future Perspectives
NGS methods, especially WES and TCR-seq are important when obtaining information from cancer genomics and treatment response
Identification of mutations (WES-data)
Identification of clonal expansion of T-cells (TCR-seq)
Identification and evaluation of combinatory treatments
IPRES + MAPKi and anti-CTLA-4
T-cell response to tumor cells after combinatory treatments
Types of tumors responsive to the treatment

18. References:
Abbas, A. K., Lichtman, A. H., & Pillai, S. (2017). Cellular and molecular immunology 9th edition. Elsevier Health Sciences. Hugo, W., Zaretsky, J. M., Sun, L., Song, C., Moreno, B. H., Hu-Lieskovan, S., ... & Seja, E. (2016). Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell, 165(1), LE, Dung T., et al. (2017) Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science, :